This invention relates generally to gene therapy. More specifically, the invention relates to suppressing immune system response to antigens expressed on an infected host cell.
The proper function of the immune system of an organism is to attack and neutralize materials which are perceived as being foreign to that organism. T-cells are one component of the immune system. T-cells can become activated to specific antigens, and function to directly destroy materials which display that antigen, and they also function to sensitize other components of the immune system to the presence of that antigen. While a properly functioning immune system is vital to the health of an organism, in some instances there is a need for the selective inhibition of an immune response to particular materials.
For example, viral vectors, such as adenovirus, are employed in genetic therapies to introduce genetic material and products into an organism. One problem encountered with the use of such viral vectors is that they can provoke an immune response in the organism. This immune response can destroy the viral vector, and those host cells which are intentionally infected by the vector, as well as therapeutic gene products produced by the action of the vector. Furthermore, immune system xe2x80x9cmemoryxe2x80x9d provides a lasting response to this vector; hence, readministration of the material will be ineffective. Therefore, there is a need for a method whereby the immune response to a selected viral vector may be blocked or destroyed. Suppression of immune response is also desirable in the instances of autoimmune disease. As is known, such disease results when the immune system of an organism inappropriately recognizes an organ or tissue of that organism as being foreign, and commences an immune response against it. If this immune response can be blocked, the autoimmune disease can be controlled. Immune suppression is also needed in those instances where organs are transplanted. Immune system suppressing drugs are sometimes employed in the foregoing situations; however, such drugs produce a generalized suppression of the immune system, which leaves a patient open to a number of infections. It would therefore be advantageous if immune response to a specific antigen could be suppressed and/or an immune suppressed zone of tissue created within an organism.
Gene therapy is limited by induction of an immune response to the virus or the gene-therapy protein product (1-4). A specific T-cell response to the viral vector usually results in the failure of re-expression of transgene (5-6). Many efforts have been made to reduce the T-cell response to the viral vector during gene therapy, including the blockade of MHC class I and II antigen, reduction of the antigenicity of the viral vector, and prevention of co-stimulation of T-cells (1,7-11).
One important mechanism for maintaining peripheral T-cell tolerance is clonal deletion of antigen-specific T-cells, which is mediated by apoptosis (12-15). Cytokine and cytokine ligand mediated apoptosis has been shown to be an important pathway for activation-induced cell death in T-cells (16-17). T-cell activation leads to upregulation of cytokine ligand and cytokine apoptosis signaling (18,19). Activated macrophages express increased levels of cytokine ligand and mediate apoptosis in the T-cells during antigen presentation, which has been thought to be a critical means of down-modulating T-cell response (20,21).
In particular, the efficiency of adenovirus-mediated gene transfer has been found to be far superior to other methods for the treatment of heart, lung, and liver disease, and is capable of producing more recombinant protein (22,23). However, the cell-mediated immune response to E1a-E3-deleted adenoviral (Ad5) vector and the limited distribution of reporter gene expression suggest that less immunogenic recombinant vectors and more homogeneous administration methods are required before Ad5 vectors can be used successfully for phenotypic modulation. Neonatal intrathymic injection of the vector was able to induce long-term LacZ expression for more than 2 months after heart injection, although neutralizing as well as anti-xcex2-Gal antibodies were detected in the sera of the animals (24). Pretreatment with the anti-TCR monoclonal antibody (mAb) H57 resulted in a significant reduction in lymphocytic infiltration and a prolongation of transgene expression (25). Studies with adenoviral vectors show that immune responses limit the efficacy and persistence of gene expression. HSVtk/ganciclovir therapy was more effective in nude rats and immunosuppressed Fischer rats than in immunocompetent Fischer rats (26). The immune response against adenovirally transduced cells limits the efficacy of the HSVtk/ganciclovir system and that immunosuppression appears to be a useful adjunct. Adenoviral transgene expression was transient in the thymus of immunocompetent mice but persistent in CD8xe2x88x92 T-cell-deficient and severe combined immunodeficiency (SCID) mice, implicating a role for cytotoxic T lymphocytes in viral clearance (27). Intrathymic transplantation of syngeneic pancreatic islet cells infected with adenovirus impaired the normal antiviral cytotoxic T-lymphocyte response and prolonged hepatic transgene expression after an intravenous challenge with adenovirus.
Ad5 vector expressing the lacZ transgene, upon delivery intra-articularly (5xc3x97108 p.f.u.), lacZ expression was observed in the articular synovium for at least 14 days. Anti-T-cell mAbs may be useful in inhibiting this immune response. Improved cell lines allow propagation of Ad with less genetic material, which decreases the antigenicity (28). The biologic efficacy and safety profile of second-generation adenovirus for CFTR gene was evaluated after transfer to baboon lung. This second-generation virus is deleted of E1 and contains a temperature-sensitive mutation in the E2a gene, which encodes a defective DNA-binding protein. Using a second-generation adenovirus, recombinant gene stability was prolonged and associated with a diminished level of perivascular inflammation as compared to first-generation vectors (29). These data suggest that second-generation adenoviral vectors provide an improved gene delivery vehicle and are useful in gene therapy for diseases such as cystic fibrosis.
Previous attempts to inhibit the immune response to adenovirus vector or transgenic products have all limited the utility of transgenic therapies. One technique of pre-toleration of the adenovirus is to induce neonatal toleration (30). Intratracheal administration of E1 deleted adenovirus within three days of birth resulted in transgene expression for over 6 months in cotton rats. Readministration of virus into 8 to 10 week old animals resulted in low levels of neutralizing antibodies. Later there was a T-cell response which correlated with existence of the transgene from the vector administered at birth, and also the eventual development of neutralizing antibodies (30). Neonatal administration of E1 deleted adenovirus to the small intestines also prolonged gene expression and decreased inflammatory response. Other investigators have used oral tolerance in rats to prolong gene expression and enable repeated injections lasting 100 days along with markedly inhibited lymphocyte response (31). The present invention for tolerance induction has the advantage that it does not require neonatal administration of the adenovirus.
Another mechanism of tolerance is the use of immune privileged sites. This tolerance makes use of the natural occurrence of immune privileged sites which has more recently been thought to be due to production of Fas ligand in subsequent killing of T-cells that may develop and react with antigens within these sites. Installation of adenovirus into these sites results in tolerance to adenovirus and its transgene product. This has been tested using E1 deleted adenovirus injected into this subretinal space which resulted in minimal cellular and humeral immune response (32). The pancreatic islet may also be an immune privileged sites since murine pancreatic islets injected ex-vivo with Ad5 resulted in high level of beta galactosidase for at least 20 weeks after re-implantation (33). Adenovirus mediated gene transfer in adult mouse islets does not impair insulin secretion by the islets (34). Ad lacZ injected subretinally resulted in prolonged gene expression, which was equivalent to that observed in either nude mice or after treatment with CTLA4Ig (8). The present invention is more widely applicable since transgene expression is not restricted to immune privileged sites.
In the instant invention antigen presenting cells (APCs) that express apoptosis inducing ligands and processed viral vector antigens are utilized to directly induce apoptosis of T-cells expressing the ligand receptor resulting in vector-specific T-cell tolerance. High levels of ligand and vector antigens are induced in APCs by co-infection. In the case of Fas ligand (FasL) as the cytokine and adenovirus vector co-infection with AdLoxpFasL+AxCANCre, pre-treatment of recipient mice with the adenovirus-infected APCs that express Fas ligand resulted in induction of T-cell tolerance to the adenovirus. The decreased T-cell response to the viral vector is demonstrated by decreased cytokine production, decreased cytotoxic T-cell response, inhibition of clonal expansion of CD3+T-cells, and prolonged the expression of a marker transgene. Induction of T-cell tolerance to adenovirus requires expression of FasL on the APCs, and does not occur with adenovirus infected control APCs. T-cell tolerance also requires expression of Fas on the T-cells of recipient mice, since 1pr/1pr mice are not tolerized. The T-cell tolerance is virus antigen-specific as there is normal T-cell response to mouse cytomegalovirus (CMV) in tolerized mice. These results indicate that pre-tolerization with syngeneic APCs co-infected with AdLoxpFasL +AxCANCre is a novel immunointervention strategy for tolerance induction to adenovirus gene therapy.
The instant invention includes a method for promoting immunotolerance in a host to a gene therapy vector, including transfecting a host cell with the vector, such that the vector expresses a transgene, an antigen and a ligand. Expression of the ligand induces apoptosis in a T-cell that is raised against the antigen.
The instant invention also includes a method for creating an immune privileged site in a tissue of an organism, the method including providing a gene therapy vector encoding and capable of expressing a ligand, a transgene and an antigen and infecting cells of the tissue with the vector. The expression of the ligand in the tissue thereby induces apoptosis in T-cells raised against the ligand so as to confer specific immunity to infected cells.
The instant invention also teaches a gene therapy viral vector that includes a transgene, an apoptosis ligand gene and a gene expression control means for directing product synthesis of said transgene and said ligand gene. In addition, the use of such a vector for a gene therapy application is detailed.
The instant invention also discloses a gene therapy viral vector including a transgene, a viral vector gene that is expressed as an antigen on an infected host cell, a functional equivalent of a Fas ligand gene and a gene expression control means for directing product synthesis of said transgene and said Fas ligand gene.